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Sample GSM5795711

Query DataSets for GSM5795711

Status

Public on Oct 06, 2023

Title

siMETTL3_2_input

Sample type

SRA

Source name

human macrophages

Organism

Homo sapiens

Characteristics

cell type: THP1 monocyte cell line -derived macrophages
treatment: transfected with METTL3 siRNA pools for 48 h.
rip antibody: none

Treatment protocol

THP1-derived macrophages were transfected with METTL3 siRNA pools or control siRNA for 48 h. Then, macrophages were stimulated with IL-4 (20 ng/ml) for 24 h.

Growth protocol

Human THP1 cell lines (purchased from the American Type Culture Collection (ATCC), USA) were maintained in RPMI-1640 medium, 10% heat-inactivated FBS. To generate macrophage-like cells, THP-1 cell were seeded along with 200 nM PMA for 48 h. Cells were incubated at 37 °C with 5% CO2.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using RNeasy Micro Kit (Cat#74004, Qiagen)following the manufacturer’s instructions and checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).Qualified total RNA was further purified by RNAClean XP Kit (Cat A63987, Beckman Coulter,Inc.Kraemer Boulevard Brea, CA,USA)and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany)
We utilized input and post m6A-IP positive fraction for library construction utilizing the Illumina TrueSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Data processing

library strategy: meRIP-seq
Illumina Casava1.7 software used for basecalling.
Raw data (raw reads) of fastq format were firstly processed using the Trimmomatic software. Clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data.Meanwhile, Use the SortMeRNA software for ribosomal RNA reads removal.After discarded the rRNA reads, the remainly clean reads were mapped to the reference genome GRCh38.p12 using HISAT2 with default parameters. Unique reads with high mapping quality were retained
The m6A-enriched peaks in each m6A immunoprecipitation sample were identified by MeTDiff peak calling software with the corresponding input sample serving as control. MeTDiff was run with options (‘FRAGMENT_LENGTH=200,PEAK_CUTOFF_PVALUE = 0.05, PEAK_CUTOFF_FDR = 0.05’) for peak detection. Called peaks were annotated by intersection with gene architecture using ChIPseeker
Differential analysis for m6A-Seq identifies differences in RNA methylome in a case-control study, The differential peak were detected by MeTDiff with parameter (‘FRAGMENT_LENGTH = 200, PEAK_CUTOFF_PVALUE = 0.01,DIFF_PEAK_CUTOFF_FDR = 0.05, PEAK_CUTOFF_FDR = 0.05’), after which,differential peak were annotated by ChIPseeker
Genome_build: GRCh38.p12
Supplementary_files_format_and_content: peak text files of every group
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every input sample

Submission date

Jan 09, 2022

Last update date

Oct 06, 2023

Contact name

Xiao Han

E-mail(s)

sqhx12@126.com

Phone

02164932897

Organization name

Children’s Hospital of Fudan University